Project Summary: HIV infection can damage the endothelial lining of vessels, including those that form the blood-brain barrier (BBB). Methamphetamine (METH) is a widely abused psychostimulant, whose abuse is a detrimental co- morbidity with HIV infection. To date, research has focused on the neurotoxic effects of METH. However, in addition to causing the death of neurons, METH can induce apoptosis in a variety of cell types, including immune cells, glial cells, stem cells, and brain microvascular endothelial cells (BMVECs). Both in vitro and in vivo studies have determined that METH disrupts the integrity of the BBB by decreasing expression of structural proteins and increasing permeability. METH?s effects appear to be dose dependent. At neurotoxic levels, METH induces apoptosis of the BMVECs that line the BBB, causing BBB damage. At a lower dose, METH inhibits proliferation and differentiation of endothelial progenitor cells (EPCs), which could negatively affect the endogenous recovery process of the damaged endothelium. While studies have shown that METH damages BMVECs, few, if any, studies have examined the effects of METH on the repair and restoration of an already-damaged BBB such as HIV-induced BBB damage. This research deficit is primarily due to the lack of an adequate experimental animal model. We have recently developed an intermedilysin (ILY) cell ablation model to mimic the endothelial injury induced in the pathological conditions. In our protocol, an endothelial cell- specific partial ablation model is first generated using a non-lethal dose of ILY in HIV-1 transgenic mice (HIVTg26 mice). EPCs prepared from commercially available ?enhanced? green fluorescent protein (EGFP) transgenic mice can then be transplanted into that model. The EPCs differentiate into endothelial cells to restore the partially damaged endothelial cell lining and recover the integrity of the BBB. The location of transplanted EPCs and progression of BBB recovery can be monitored by the EGFP upon excitation light. We propose to use our endothelial cell ablation model in HIVTg26 mice to mimic the damaged endothelium that occurs in disease states such as HIV infection in order to test our working hypothesis that the repair of the endothelial cell lining of a damaged BBB by EPCs is impeded by METH and worsened in the presence of HIV viral proteins. Our specific aims are to investigate: 1) the effects of METH on the repair and restoration of the damaged endothelial cell lining of the BBB in the presence of HIV viral proteins in vivo (Aim 1); and 2) the molecular mechanisms underlying METH?s effects on EPC restoration of BBB integrity in the presence of HIV viral proteins ex vivo (Aim 2). The findings from this highly significant and clinically relevant exploratory project will shift the focus from METH?s damaging effects on the endothelial cell lining to METH?s detrimental actions on endothelial cell recovery, particularly with regard to the repair and restoration of a compromised BBB as a result of HIV infection.